In recent years recombinant DNA technology has made possible the large scale production of proteins. Several methods for the solubilization and naturation of somatotropin protein have been the subject of U.S. Patents. For example U.S. Pat. No. 4,511,503 discloses a typical scheme for recovering proteins from refractile bodies. Refractile bodies are insoluble granules of aggregated denatured somatotropin located in the cytoplasm of the Escherichia coli (E. coli) cell which are visible as bright spots under a phase contrast microscope. The refractile bodies are caused by the over production of somatotropin as a result of genetic manipulation of the E. coli plasmid DNA. The refractile bodies are often treated with a strong denaturant or chaotropic agent which causes the improperly folded molecules to unfold and become soluble. The protein must then be "renatured." Properly natured monomeric somatotropin is the goal. The refractile bodies cannot be used without this unfolding and refolding because they are biologically inactive in the refractile state. The most commonly employed strong denaturant in schemes of this type has been guanidine hydrochloride.
Other methods have involved other chaotropic agents such as sodium dodecyl sulphate (SDS) (e.g. U.S. Pat. No. 4,677,196), or weak denaturants such as urea (e.g. U.S. Pat. No. 4,731,440).
Each of the methods of solubilization and naturation of somatotropin have had problems. Guanidine hydrochloride is very expensive and must be replaced for the naturation process to occur. SDS is a highly effective denaturant and much less expensive than guanidine hydrochloride, but SDS binds to the denatured protein much more tightly making its complete removal from the protein problematic and concurrently increasing processing costs. Urea is usually used as a weaker denaturant or chaotropic agent. But even methods using urea have had problems such as contamination of the final product and handling, storage and waste treatment problems.
In addition to the other problems of conventional methods, properly natured monomer is not the only product. Somatotropin monomer is the smallest unit of protein that still retains all of the properties and biological activity of somatotropin. Typically somatotropin monomer consists of approximately 191 amino acid residues and has a molecular weight of roughly 22,000 daltons. The monomeric molecule is neither covalently linked to nor non-covalently associated with other similar molecules.
Somatotropin dimer consists of two monomer molecules which are either covalently linked, e.g. through intermolecular disulfide bonds, or non-covalently associated with one another. The dimer molecule consists of double the number of amino acid residues and double the molecular weight of a monomeric molecule.
Unfortunately employing conventional methods, some dimer is formed, as well as higher molecular weight protein molecules. Only the monomer and not the dimer is biologically useful.
Therefore what is needed in the art is a commercially feasible method for the solubilization and naturation of somatotropin which produces good yield of monomer, without excess dimer and without the use of chaotropic agents.